Extended Data Fig. 5: PP6R3 controls phosphorylation of TRF2 at Ser365 or Ser367.
From: CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle
HEK 293 cells expressing wild-type Myc–TRF2 were transfected with a non-targeting control siRNA or siRNAs against protein phosphatase regulatory subunits (a) or catalytic subunits (b). Cells were collected, and whole-cell extracts were immunoprecipitated with anti-RTEL1 antibody. Immunocomplexes were resolved by SDS–PAGE and analysed by western blotting as indicated. c, HEK 293 cells (c) and Trf2F/− MEFs (d) expressing Myc-tagged wild-type TRF2 were transfected with control siRNA or siRNA targeting PP4R2 or PP6R3 (Pp4r2 or Pp6r3 for MEFs). Whole-cell extracts were immunoprecipitated with anti-TRF2 antibody, and immunocomplexes were resolved by SDS–PAGE and analysed for human phospho-TRF2 (pS365 TRF2; left panel in c) or mouse phospho-TRF2 (pS367 TRF2; left panel in d). e, Top, frequency of telomere loss and telomere fragility per metaphase in Rtel1F/F MEFs transfected with control siRNA or with Pp4r2 or Pp6r3 siRNA (n = 58 (NTC), n = 57 (Pp4r2), and n = 55 (Pp6r3) of analysed metaphases). Efficiency of siRNA knockdown was determined by western blotting with PP6R3 and PP4R2 antibodies as indicated. Data are mean ± s.e.m. P values determined by one-way ANOVA. Bottom, representative images of the telomere FISH experiments. The arrowheads show loss of telomere signal. Red, telomere PNA FISH; blue, DAPI. f, Phi29-dependent telomere circles (top) detected in cells as indicated in e. The extent of [32P] incorporation was quantified (bottom) from the autoradiographs, and the level of [32P] incorporation by cells transfected with control siRNA was arbitrarily assigned a value of 100%. Data are mean ± s.d. and from two independent experiments. P values determined by one-way ANOVA. In a–f, the experiments were independently repeated at least twice with similar results.
